The wild-type p53-induced phosphatase Wip1 (PPM1D) is a member of the serine/threonine protein phosphatase 2C (PP2C) family. Although Wip1 is expressed at low levels in most normal cells, its transcription is induced by p53 after exposure of cells to DNA damage-inducing agents, such as ionizing radiation (IR) or ultraviolet (UV) light. Resulting from genomic amplification, transcriptional dysregulation, or impaired protein degradation, the Wip1 protein is frequently overexpressed in several human cancers, and this icreased expression is generally associated with a worse prognosis. Cellular studies have shown that overexpression of Wip1 compromises the functioning of several tumor suppressors, while other studies have demonstrated that mice lacking Wip1 expression are resistant to tumorigenesis. Our current research on Wip1 is focused on understanding its regulation and functions, identifying its functional targets and performing high-throughput screens to identify specific inhibitors of Wip1 phosphatase activity. Ubiquitous Wip1 deletion in mice affects the immune system, organismal metabolism and the tumor micro-environment, all of which may affect tumorigenesis in the organ of interest. To overcome these limitations, we have generated a conditional knock-out mouse in which the inactivating deletion of Ppm1d exon 3 can be restricted to a single tissue through tissue-specific expression of Cre recombinase or implemented at a specified time through inducible expression of Cre recombinase. These conditional Wip1 knock out mice are proving to be useful in a variety of models of tumorigenesis. Recently, in collaboration with Dr. Oleg Demidov, University of Burgundy, France, we have been developing model systems to investigate the effects of deletion of Wip1 at various times. These model systems will allow us to investigate the roles of Wip1 in tumor initiation, tumor progression and metastasis. Wip1 dephosphorylates serine and threonine residues within pTXpY and pTQ/pSQ motifs. Many of the known pTQ/pSQ substrates are phosphorylated by ATM. We have undertaken a quantitative phosphoproteomic analysis to provide a characterization of the substrate specificity of the Wip1 phosphatase. In our experiments, we have used the stable isotope labeling with amino acids in cell culture (SILAC) approach to label cells in culture for quantitation of the relative change in phosphorylation sites following cellular stress under conditions of high or low Wip1 activity. These studies identify sites of phosphorylation that are affected by Wip1 activity. Preliminary experiments identified more than 800 phosphorylation sites of which nearly 10% are affected by modulation of Wip1 activity. Among the proteins containing phosphorylation sites affected by Wip1 activity are several transcription factors and kinases. These studies provide critical insights into Wip1 substrates and function. PP2C serine/threonine protein phosphatases are critical regulators of stress responses and are distinguished by a requirement for supplementation with millimolar concentrations of Mg2+ or Mn2+ ions for activity in vitro. The crystal structure of human PPM1A, the first PPM structure determined, showed two tightly bound Mn2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. We recently determined the crystal structure of the catalytic domain of human PPM1A, PPM1Acat, complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK, a physiological substrate of PPM1A. The structure revealed three metal ions in the active site confirming the presence of a third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational analyses demonstrated that the complex has a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain and that the position of the substrate in the active site allows solvent access to the labile third metal binding site. Enzyme kinetics of PPM1Acat toward a phosphopeptide substrate showed a random order, bi substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. These results support the idea that conformational stabilization of the Flap, which is critical for specific substrate recognition, may be coupled to catalytic activity through the binding of the third metal ion. These fundamental characteristics of the catalytic mechanism are likely to be shared by PPM1A and Wip1, as the implicated residues are highly conserved. Wip1 is amplified or overexpressed in numerous human cancers including breast cancer, ovarian clear cell carcinoma, gastric cancer, pancreatic adenocarcinoma, medulloblastoma, and neuroblastoma. Thus, developing inhibitors of Wip1 activity may be beneficial in the treatment of a number of human cancers. Based on biochemical and biophysical screening of small molecule and DNA encoded compound libraries, an allosteric inhibitor of Wip1, GSK2830371, was recently reported (Gilmartin et al. 2014). However, although GSK2830371 is a selective and potent inhibitor of Wip1 activity in cells due to its interaction with the Flap of Wip1, it does not exhibit favorable pharmacokinetics. As the Flap subdomain is the key structural linkage between the activity and the substrate selectivity of PP2C phosphatases, we carried out structure activity relationship (SAR) studies on Flap subdomain based inhibitors identified in the biochemical high throughput screen (HTS) reported by Gilmartin et al., as very limited SAR information was available for these classes of compounds. Our thorough SAR study identified new analogues that exhibited good affinity and bioavailability and provided critical insights into designing new compounds that target the Flap subdomain of Wip1. In addition, we are pursuing several strategies to provide structural information about the Wip1 catalytic site and the mechanism of Wip1 inhibitors. We are also developing a high throughput screening of Wip1 activity utilizing a p53 peptide containing p53pS15 and are collaborating with the National Center for Advancing Translational Sciences (NCATS).